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Engineering yeast for improved wine aroma: Release of 3-1
Mercaptohexan-1-ol during fermentation through overexpression 2
of a yeast gene, STR3 3
4
Sylvester Holt1,2†
, Antonio G. Cordente3†
, Simon J. Williams2, Dimitra L. Capone
3, 5
Wanphen Jitjaroen4, Ian R. Menz
2, Chris Curtin
3* and Peter A. Anderson
2 6
7
University of Copenhagen, Copenhagen, Denmark, 1 School of Biological Sciences, Flinders 8
University, Adelaide, SA, Australia, 2 The Australian Wine Research Institute, Adelaide, SA, 9
Australia, 3 Rajamangala University of Technology Lanna, Lampang, Thailand
4 10
11
*Corresponding author. Mailing address: The Australian Wine Research Institute, Wine 12
Innovation Cluster, P.O. Box 197, Urrbrae SA 5064, Australia. Phone: 61-883036645. Fax: 13
61-883036601. E-mail: [email protected]. 14
†Both authors contributed equally to the manuscript. 15
16
Running title: Release of 3MH by overexpression of yeast STR3 during fermentation 17
18
Keywords: cystathionine β-lyase, volatile thiols, pyridoxal-5’-phosphate, wine yeast, wine 19
aroma, carbon-sulfur lyase, tryptophanase 20
21
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.03009-10 AEM Accepts, published online ahead of print on 8 April 2011
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ABSTRACT 22
Sulfur containing aroma compounds are key contributors to the flavor of a 23
diverse range of foods and beverages. The tropical fruit characters of Sauvignon Blanc 24
wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3MH), 25
3-mercaptohexan-1-ol-acetate and 4-mercapto-4-methylpentan-2-one (4MMP). These 26
volatile thiols are found in small amounts in grape juice and are formed from non-27
volatile cysteinylated precursors during fermentation. In this study, we overexpressed a 28
yeast gene, STR3, which led to an increase in 3MH release during fermentation of a 29
Sauvignon Blanc juice. Characterization of the enzymatic properties of Str3p confirmed 30
it to be a pyridoxal-5’-phosphate dependent cystathionine β-lyase, and we demonstrated 31
that this enzyme was able to cleave the cysteinylated precursors of 3MH and 4MMP, to 32
release the free thiols. These data provide direct evidence for a yeast enzyme able to 33
release aromatic thiols in-vitro, that can be applied in the development of self-cloned 34
yeast to enhance wine flavor. 35 on January 14, 2020 by guesthttp://aem
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INTRODUCTION 37
Aromatic thiols are potent aroma compounds, with a sensory perception threshold 38
range in the parts per trillion. They are found in a wide range of foods (38), including animal 39
products such as yoghurt and cheese, fruits and vegetables, tea, coffee and alcoholic 40
beverages (2, 5, 32, 34, 37). Of particular interest in wine fermentation are the aromatic 41
volatile thiols, 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH), 42
and 3-mercaptohexyl acetate. These compounds impart flavors such as “grapefruit”, 43
“passionfruit” and “boxwood” and are major contributors to the varietal character of Vitis 44
vinifera L. var. Sauvignon Blanc white wines (17). Cysteine-S-conjugated bound forms of 45
these free aromatic thiols are present in grape juice and are transformed into flavor active 46
thiols during fermentation by yeast (29). The carbon-sulfur (CS) β-lyase activity that is 47
necessary for transformation of cysteine-S-conjugated forms of 3MH and 4MMP into free 48
thiols was first inferred from cell-free extracts of Eubacterium limosum and Allium sativum 49
(33). Since then, the potential for enhanced thiol release in grape juice has been demonstrated 50
by the constitutive expression of the Escherichia coli tnaA gene, a tryptophanase with strong 51
CS β-lyase activity (28). Thus far, there is no direct evidence of such a yeast-derived 52
enzymatic activity releasing aromatic thiols under oenological conditions, although some 53
candidate genes have been suggested based on a gene deletion approach (15, 30). The release 54
of aromatic thiols by other microorganisms has been linked to the activity of cystathionine β- 55
and γ-lyases. For example; Lactobacillus casei and Lactobacillus lactis in cheese production 56
(16, 20), Staphylococcus haemolyticus in the release of human body odor (36), and 57
Streptococcus anginosus in mouth malodor (41). 58
Apart from its potential role in aromatic thiol release, cystathionine β-lyase (CBL, EC 59
4.4.1.8) is involved in the biosynthesis of methionine. CBLs catalyze the conversion of 60
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cystathionine into homocysteine in an α,β-elimination reaction, which in a later step is 61
converted to methionine (31). This reaction is dependent on the co-factor, pyridoxal-5'-62
phosphate (PLP), a derivative of vitamin B6. Some bacterial CBLs have been extensively 63
characterized since the methionine biosynthetic pathway is absent in mammals, and thus is an 64
important antibiotic target (8, 9). In contrast, the enzymatic characterization of eukaryotic 65
CBLs is more limited, with the exception of two plant CBLs from Arabidopsis thaliana (22) 66
and Spinacia oleracea chloroplasts (27), for which the A. thaliana crystal structure has been 67
solved (6). In yeasts, the Schizosaccharomyces pombe STR3 gene product has been shown to 68
have activity towards cystathionine (9). Such activity can be attributed to the Saccharomyces 69
cerevisiae STR3 homologue, YGL184C, as a strain containing a null mutant was unable to 70
grow on glutathione or cystathionine as a sole sulfur source (12). 71
In this study, we purified the S. cerevisiae gene product, Str3p, and confirmed its 72
activity as a CBL. Furthermore we provide direct evidence that a purified form of this yeast 73
enzyme has activity towards cysteine-S-conjugated precursors of the aromatic thiols 3MH 74
and 4MMP. When the STR3 gene is overexpressed in a commercial wine yeast used to 75
ferment S. Blanc grape must, an increase in 3MH release is detected. These data provide the 76
basis on which a self-cloning approach could be adopted to improve the sensory properties of 77
white wine. 78
MATERIALS AND METHODS 79
Similarities to the primary protein sequence of Str3p were identified using LALIGN 80
(http://www.ch.embnet.org/software/LALIGN_form.html). 81
Chemicals. All reagents were of analytical grade and purchased either from Amresco 82
or Sigma-Aldrich unless otherwise stated. KH2PO4, K2HPO4, KCl and glycerol were obtained 83
from Chem-Supply (Adelaide, Australia). 4MMP and S-4-(4-methylpentan-2-one)-L-cysteine 84
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(Cys-4MMP) were synthesized as described by Howell et al. (14), and [2H10]-4MMP and 85
3MH were synthesized as described by Kotseridis et al. (18). The synthesis of [2H10]-3MH is 86
described in Pardon et al. (21). S-3-(hexan-1-ol)-L-cysteine (Cys-3MH), as a mixture of 87
diastereoisomers, was prepared using the procedure of Wakabayashi et al. (39). 88
Microbial strains, media and culture conditions. Chemically competent E. coli 89
DH5α and BL21(DE3) cells (New England Biolabs) were used for amplification of plasmid 90
DNA and protein expression, respectively. Growth and selection was carried out in Luria-91
Bertani medium supplemented with 30 µg/ml kanamycin. Commercial wine yeast strain 92
VIN13 (Anchor Yeast, Cape Town, South Africa) was used as host strain for the expression 93
of the STR3 gene cassette. The commercial wine yeast strain EC1118 (Lallemand, Canada) 94
was used as a source of genomic DNA. 95
Yeast strains were cultivated at 30°C in either a rich medium, YPD (containing 1% 96
yeast extract, 2% peptone and 2% glucose), or a synthetic dropout medium, SCD [containing 97
2% glucose, 0.67% yeast nitrogen base without amino acids (Difco, Detroit, MI, USA)]. For 98
the selection of sulfometuron methyl (SMM) resistant yeast transformants, SCD media was 99
supplemented with 50 µg/ml SMM (Dupont, Wilmington, DE, USA) dissolved in N-N-100
dimethylformamide. Solid media contained 2% agar (Difco). 101
DNA constructs. Standard procedures for the isolation and manipulation of DNA 102
were used as described in Ausubel et al. (1). The pDLG42-CSL1 plasmid (28) served as 103
template to amplify the E. coli tryptophanase (tnaA) gene using Phusion® High-Fidelity 104
DNA Polymerase (Finzymes). For E. coli expression of proteins with a C-terminal 105
6xHistidine tag, we first cloned the tnaA gene into the pET-24 (+) vector (Merck 106
Biosciences) with the primers pET24-TnA-FWD (5'-107
TCTAGGATCCAAAATAAGGAGGAAAAACATATGAAGGATTATGTAATGGAAAAC108
TTTAAAC-3’) and pET24-TnA-REV (5'-109
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GTGCTCGAGAACTTCTTTCAGTTTTGCGGTGAAG-3’) using BamHI and XhoI 110
restriction sites (underlined in both primers). The subsequent construct, designated pET-T, 111
had an E. coli Shine Delgarno sequence (boxed) (23) to ensure efficient translation, and an 112
NdeI restriction site. The S. cerevisiae STR3 gene was amplified from genomic DNA from 113
the wine yeast strain EC1118 with primers pET24-STR3-FWD (5'-114
TCTACATATGCCGATCAAGAGATTAGATACA-3’) and pET24-STR3-REV (5'-115
GTGCTCGAGCAATTTCGAACTCTTAATATTCAATTCTGA-3’). The STR3 coding 116
region (1398 bp) was cloned using the NdeI and XhoI restriction sites (underlined in both 117
primers) in the pET-T plasmid, thus yielding the pET-STR3 construct. 118
The pDLG42-PGK1 plasmid was constructed by cloning the 1.8 kb HindIII fragment 119
released from the pHVXII plasmid, containing the phosphoglycerate kinase I gene (PGK1) 120
constitutive promoter (PGK1P) and terminator (PGK1T) cassette, into the HindIII site of the 121
yeast single-copy integrating plasmid pDLG42. This plasmid contains the ILV2 (SMR1-410) 122
marker gene, which confers resistance to SMM. To clone STR3 into the pDLG42-pGK1 123
plasmid, the gene was amplified by PCR using pET-STR3 as a template, and primers STR3-124
XhoI-FWD (5’-GACTCTCGAGATGCCGATCAAGAGATTAGATAC-3’), and STR3-125
XhoI-REV (5’-TAGCCTCGAGTTACAATTTCGAACTCTTAATATTC-3’), which were 126
engineered to introduce a XhoI restriction site (underlined in both primers). The PCR product 127
was cloned into the XhoI site of the pDLG42-PGK1 plasmid, between the PGK1P and PGK1T 128
sequences. The resulting plasmid, pDLG42-PGK1-STR3, was linearized with ApaI and 129
transformed into VIN13. Transformants were selected in SCD-SMM media, genomic DNA 130
was isolated, and the integration of the STR3 expression cassette into the genome was 131
confirmed by PCR. This transformant was designated VIN13 (STR3). The integrity of all 132
expression constructs used in this study was confirmed by DNA sequencing using the 133
Australian Genome Research Facility, Brisbane, Australia. 134
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Protein expression and purification. Transformants were grown in 250 ml Luria-135
Bertani media to the log phase (OD600 = 0.6-0.8) from an overnight culture supplemented 136
with 30 µg/ml kanamycin. Expression of recombinant protein was induced with 1 mM IPTG 137
at 18°C. After 16 h, the bacterial cells were harvested by centrifugation and washed twice 138
with 20 mM Tris pH 7.5, 130 mM KCl, 10% (w/v) glycerol, 10 mM EDTA. About 1.5 g of 139
cell pellets were resuspended in cell lysis buffer (50 mM Tris pH 8.0, 100 mM KCl, 10% 140
(w/v) glycerol, 0.25 mM Triton X-100, 10 mM imidazole, 100 µM PLP, and protease 141
inhibitors 1 mM phenyl-methanesulfonyl fluoride and 5 mM έ-amino-n-caproic acid), and 142
lysed by four sequential passes through a pre-cooled cell disruptor (8,000-15,000 psi) 143
(EmulsiFlex-05 Homogenizer, Aventis). All subsequent steps were carried out at 4°C. The 144
lysates were centrifuged at 45,000 X g for 1 h in a Sorvall SW32 rotor and filtered through a 145
0.22 µm (Millipore) filter. The resulting cleared lysate was then exposed to Nickel-affinity 146
chromatography using a 4 ml Ni Sepharose 6 Fast Flow matrix according the manufacturer's 147
instruction (GE Lifesciences). Protein was purified by washing with 5 column volumes of 148
NiA buffer containing 20 mM Tris pH 7.4, 250 mM KCl, 100 µM PLP, and 10 mM 149
imidazole, and a subsequent wash with 5 column volumes of NiA-wash buffer containing 100 150
mM imidazole for Str3p and 70 mM for tryptophanase. Bound protein was then eluted in NiA 151
buffer containing 500 mM imidazole for Str3p and 250 mM for tryptophanase. Protein was 152
separated by electrophoresis on NuPAGE 10% Bis-Tris SDS-PAGE gels with MOPS-SDS 153
running buffer (Invitrogen) and stained with imperial stain (Thermo Scientific). The 154
molecular weight of monomeric Str3p was determined from the Prescision Plus molecular 155
weight marker (Bio-Rad). 156
Dialysis, protein concentration and estimation. Purified proteins were dialyzed 157
overnight at 4°C with continuous stirring in 1 liter buffer A (20 mM Tris pH 8.0, 500 mM 158
KCl, 20 mM EDTA, 8% (w/v) glycerol, 100 µM PLP) for Str3p, and 20 mM potassium 159
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phosphate, pH 7.0, 10 µM PLP for tryptophanase. Dialyzed enzymes were concentrated at 160
4°C with Vivaspin 15R 10,000 MWCO concentrators (Sartorius Stedim Biotech) to 10-12 161
mg/ml. Protein concentration was determined using a Bio-Rad protein assay with a standard 162
of Bovine Serum Albumin. Purified Str3p was then snap frozen and kept at -80°C. 163
Size exclusion chromatography. Size exclusion chromatography was carried out 164
with a Superdex 200HR 10/30 analytical column (GE Lifesciences) using an ÄKTA Explorer 165
100 FPLC (Pharmacia, GE Lifesciences). The flow-rate was 0.3 ml/min. Prior to sample 166
loading, columns were equilibrated with buffer A. The column was calibrated with proteins 167
of known molecular weight (Sigma-Aldrich) to produce standard curves. 168
MALDI-TOF/MS. 25 µg of purified Str3p were resolved by SDS-PAGE and 169
subjected to a trypsin digestion and Matrix Assisted Laser Desorption/Ionisation-Time Of 170
Flight (MALDI-TOF) mass spectroscopy by the Australian Proteome Analysis Facility Ltd., 171
Sydney, Australia. Data were submitted to the database search program, Mascot (Matrix 172
Science Ltd, London UK). 173
Carbon-sulfur lyase assay. Reactions were carried out in a total volume of 1 ml 174
containing a final concentration of 2 µg/ml of Str3p, 50 mM of the reaction phosphate buffer 175
pH 8.5, 20 µM PLP, 1 mM EDTA, and 2 mM of the sulfur-containing amino acid substrate. 176
Reactions were incubated for 1 h at 37°C and kept frozen until assayed for CS lyase activity. 177
For pH optimum tests, the reaction buffer contained 50 mM MES, 50 mM BTP and 50 mM 178
CAPS and, depending on the target pH (5.5-11), it was titrated with either KOH or HCl. 179
Experiments were performed in triplicate. 180
The formation of the α-ketoacids pyruvate (indicator of β-lyase activity) and α-181
ketobutyrate (indicator of γ-lyase activity) was determined by HPLC using a HPX-87H 182
Aminex ion exchange column (Bio-Rad), and an Agilent Technologies 1200 series liquid 183
chromatograph. The operating conditions were as follows: a flow rate of 0.5 ml/min, 65°C, 184
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and detection at 210 nm. The mobile phase was 5 mM H2SO4. Pyruvate and α-ketobutyrate 185
(Sigma-Aldrich) were used as standards. For the negative control, a cleared lysate from BL21 186
(DE3) cells transformed with an empty pET vector was treated in an identical way to cells 187
containing pET-STR3. For kinetic analysis we used Ellman’s reagent [5,5’-dithiobis-(2-188
nitrobenzoic acid)] to quantify free thiol groups with absorbance measured at 412 nm. 189
Michaelis-Menten kinetics. Reactions were carried out as described above with 2 190
µg/ml purified Str3p. The data from three individual experiments were pooled and fitted to 191
the Michaelis-Menten equation using PRISM (ver. 5) with R2 values of 0.91 and 0.94 for L-192
cystathionine and L-djenkolate, respectively. For calculation of the catalytic turnover and 193
catalytic efficiency we used the assumption that Str3p was purified to homogeneity. 194
Enzymatic reactions with Cys-4MMP and Cys-3MH. Cleavage of Cys-4MMP and 195
Cys-3MH were done in a total volume of 1.3 ml. E. coli tryptophanase was included as a 196
positive control. For negative controls; reactions were performed with Str3p that was heat 197
inactivated for 2 minutes at 95°C, and using protein eluted from Nickel affinity 198
chromatography and extracted from induced E. coli cells transformed with an empty pET-24 199
(+) vector. Indistinguishable concentrations of free thiol were detected in both negative 200
controls. The conditions of the reactions were as follows: 31 µg/ml enzyme, 50 mM 201
phosphate buffer pH 7.0 or 7.5, 20 µM PLP and 1 mM EDTA with 0.25 mM or 2 mM 202
substrate, incubated at 28°C for 1 h and kept at 4°C until assayed by headspace GC/MS. 203
Wine fermentation. YPD was inoculated with strains VIN13, VIN13 (STR3) and 204
VIN13 (CSL1). Yeast starter cultures were made in autoclaved grape juice, and incubated for 205
48 h at 28°C to stationary phase. Frozen 2007 S. Blanc clarified juice (obtained from the 206
Adelaide Hills, Australia) was thawed, thoroughly mixed, and filter-sterilized using a 207
VacuCap 60PF filter unit (0.8/0.2 µm pore diameter, Pall Life Sciences). 208
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The basic chemicals parameters of the juice were 198 g/l sugar, 460 mg/l yeast 209
assimilable nitrogen and pH 3.2. A volume of 200 ml of the juice were transferred to 250 ml 210
fermentation flasks with airlocks, and the juice was inoculated at a density of 1x106 cells/ml 211
from the starter cultures. The wines were fermented in triplicate at 18°C for 15 days, and then 212
cold-stabilized at 4°C. The wines were then racked, and kept in 100 ml glass reagent bottles 213
at 4°C until analysis. The concentrations of sugars, ethanol, glycerol, acetic acid, malic acid, 214
tartaric acid and succinic acid were measured by HPLC using a Bio-Rad HPX-87H column as 215
described above for quantitation of α-ketoacids. Low molecular weight sulfur compounds 216
that are known “off-odors” were quantified by gas chromatography coupled with sulfur 217
chemiluminescence detection GC/SCD (26). 218
Headspace GC/MS analysis. An aliquote of 1 ml of the enzymatic reactions (or 219
diluted tryptophanase reactions) was assayed in a total volume of 5 ml containing 220
approximately 20 mg EDTA and 2 g NaCl, in a 20 ml solid phase micro extraction vial with a 221
magnetic crimp cap (Gerstel, Baltimore, MD, USA). A solution containing a mixture of 222
deuterated standards of [2H10]-4MMP (9.88 µg/ml) and [2H10]-3MH (14.32 µg/ml) in ethanol 223
was added using a glass syringe (SGE, Grace Davison Discovery Sciences, Rowville, Vic, 224
Australia) to each of the samples. The instrumental conditions were performed as described 225
in Swiegers et al. (28) with the following modification: the autosampler was fitted with an 226
automated 2 cm Divinylbenzene/Carboxen/Polydimethylsiloxane solid phase micro 227
extraction fibre (Supelco, Bellefonte, PA, USA). For quantification, mass spectra were 228
recorded in the selective ion monitoring mode. The ions monitored were: m/z 81, 96, 108, 229
141 and 142 for [2H10]-4MMP; 55, 75, 89, 99 and 132 for 4MMP; 60, 62, 92, 109 and 144 for 230
[2H10]-3MH; 55, 82, 100 and 134 for 3MH. Selected fragment ions were monitored for 20 ms 231
each. The underlined ion for each compound was the ion typically used for quantitation, 232
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having the best signal : noise ratio and the least interference from other components. The 233
other ions were used as qualifiers. 234
Analysis of 3MH at wine like concentrations was carried out using a newly developed 235
method (D. L. Capone, M. Sefton, and D. Jeffery, submitted for publication). This method 236
involves multiple liquid extractions, followed by derivitization and uses GC/MS in selective 237
ion monitoring for detection. 238
RNA purification. Approximately 1x107 yeast cells were harvested from 239
fermentations by centrifugation, prepared in RNALater (Ambion) and kept at -80°C until 240
analysis. The cells were washed by resuspension in 300 µl ice-cold nuclease-free H2O and 241
centrifuged at 1,500 X g, 4°C. The cells were then resuspended in 100 µl Zymolyase buffer 242
(50 mM Tris-HCl pH 7.5, 1 M sorbitol and 10 mM MgCl2) containing 30 mM DTT, and 243
incubated for 15 min at room temperature to reduce any disulfide bonds. Zymolyase 20T 244
from Arthrobacter luteus (MP Biomedicals, Aurora, OH) (20 Units) was added to Zymolyase 245
buffer containing 1 mM DTT and incubated at 30°C for 40 min. 246
RNA was isolated using the PureLink™ RNA mini Kit (Invitrogen) according to 247
manufacturer’s instructions. Briefly, lysis was carried by adding 200 µl lysis buffer 248
containing 1% β-mercaptoethanol, followed by centrifugation at 16,000 X g for 2 min. Equal 249
volume of 99% ethanol was added to the supernatant followed by thorough vortexing. The 250
resulting lysate (500 µl) was transferred to a PureLink RNA mini spin column and RNA was 251
isolated according to manufacturer’s instruction. Purified RNA was treated with 1 Unit of 252
Amplification grade DNase I (Invitrogen) for 15 min at room temperature. The reactions 253
were stopped by adding 5 mM EDTA and incubation at 65°C for 5 min. A typical yield of 254
500 ng of total RNA was obtained as quantified by the QUBIT™ quantification platform 255
using Quant-iT™ RNA assay Kit and standards (Invitrogen). 256
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Reverse transcription. cDNA was synthesized from 100 ng of total RNA using an 257
oligo-dT20 primer and a AffinityScript QPCR cDNA synthesis Kit (Stratagene, Agilent 258
Technologies). All steps in cDNA synthesis followed the manufacturer’s instructions. 259
qPCR. Quantitative PCR was performed using the Bio-Rad CFX96™ real time 260
detection system with Brilliant II SYBR Green reagent (Agilent Technologies) and cDNA 261
made from 2.5 ng total RNA in a volume of 25 µl. To quantify the transcript level of the 262
STR3 gene, data was normalised using ACT1 as a reference transcript. Primers for STR3 263
(STR3.FWD: 5’- TCAAACCTACCAGAACAAACAAG-3’, STR3.REV:5’- 264
CGTCACAGCCCATATACTCAG-3’) and ACT1 (Act.FWD: 5’- 265
GCCAAGATAGAACCACCAATCC-3’, Act.REV:5’-CTGATGTCGATGTCCGTAAGG-266
3’) were validated with efficiencies of 99.4% and 100.6%, respectively. Ct values were 267
obtained from duplicate fermentations, and STR3 expression was normalised against the Actin 268
reference gene by the 2-∆∆Ct method and expressed relative to the native promoter at day 5. 269
RESULTS 270
Analysis of the S. cerevisiae cystathionine β-lyase STR3 sequence. The STR3 gene 271
from the diploid commercial wine yeast, EC1118, displayed three heterozygous allelic 272
variants, G244A, A411G, and T633C, when compared with the S288c haploid reference 273
strain. Of these, only the G244A variant results in an amino acid change in the protein 274
sequence (A82T). This variant is not present in the recently published whole genome shotgun 275
sequence of the EC1118 strain (4), assembled as a pseudohaploid due to the low rate of 276
heterozygosity observed in this strain. The STR3 gene used in our study contained both 277
G244A and T633C variants and was submitted to GenBank with the accession number, 278
HQ008776. Based on its primary amino acid sequence, Str3p is highly conserved among 279
other budding yeasts with identities of 66% for Lachancea thermotolerans, and 51% for 280
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Clavispora lusitaniae. It also displays 44% identity to the fission yeast S. pombe STR3 281
homologue, 40% identity to an A. thaliana CBL, and 29% identity to the E. coli CBL 282
encoded by metC. The Str3p protein sequence diverges from E. coli tryptophanase, both of 283
which belong to the large group of aspartate aminotransferase fold type I enzymes (24). 284
Purification of S. cerevisiae Str3p. We expressed recombinant Str3p in E. coli and 285
purified the protein in the presence of its co-factor PLP, using Ni-NTA chromatography to 286
capture the C-terminal 6xHistidine tagged protein (Fig. 1). Monomeric recombinant Str3p has 287
a predicted molecular size of 53kDa, and migrated at approximately 52 kDa on SDS-PAGE. 288
A yield of 40 mg of pure protein per liter of culture was typically obtained. The buffer 289
exchange conditions to stabilise the isolated protein and maintain its enzymatic activity 290
included 500 mM KCl and 20 mM EDTA. By including glycerol in the buffer, less than 6% 291
of the activity was lost in a freeze-thaw cycle, compared to the 56% loss in the presence of 292
imidazole. MALDI-TOF Mass Spectrometry was performed on purified Str3p and its identity 293
as the STR3-encoded protein was confirmed (E=7.9e-68). 294
The size of native purified Str3p was investigated by size exclusion chromatography. 295
A protein peak with CBL activity eluted at 13.2 ml that corresponds to a molecular weight of 296
195 kDa (data not shown). This indicates that S. cerevisiae Str3p purified from E. coli forms 297
a stable homotetramer, a finding consistent with other CBLs (7, 22, 27). PLP-enzymes with 298
co-factor bound to the active site show a characteristic absorbance at 420-435 nm (10, 40). 299
We observed absorption at 428 nm together with the protein peak, suggesting that the protein 300
was associated with PLP and purified as the holoenzyme. 301
Kinetic properties of purified Str3p. The effect of pH on recombinant Str3p activity 302
was investigated using its physiological substrate, L-cystathionine. Enzyme activity was 303
measured by the formation of pyruvate, one of the end-products of the α,β-elimination (β-304
lyase) reaction. The recombinant enzyme displayed a bell-shaped pH-rate profile, with an 305
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optimum activity at pH 8.75 (Fig. 2), and no significant activity was observed below pH 7 or 306
above pH 10.5. 307
Several sulfur-containing amino acids were assayed as potential substrates for the 308
Str3p enzyme (Table 1). The non-protein amino acid, L-djenkolate, was the most effective 309
substrate for Str3p, preferred even above its physiological substrate, L-cystathionine. To a 310
lesser extent, L-cystine, S-methyl-L-cysteine, and S-ethyl-L-cysteine, acted as substrates and 311
a residual activity was also detected using L-cysteine as a substrate. No α-ketobutyrate was 312
detected with any of the substrates susceptible for γ-lyase activity, confirming that Str3p has 313
only β-lyase activity. Reactions involving recombinant Str3p with the two substrates, L-314
cystathionine and L-djenkolate, obeyed Michaelis-Menten kinetics. The catalytic turnover 315
(Kcat) of Str3p was slightly higher for L-djenkolate than for L-cystathionine (1.27 vs. 0.91 s-316
1), while a 2-fold binding preference for L-cystathionine compared to L-djenkolate was 317
observed (Km 96 vs. 178 µM). Consequently, the catalytic efficiency, Kcat/Km, was higher for 318
L-cystathionine than for L-djenkolate. 319
Enzymatic release of 3MH and 4MMP from their cysteine-S-conjugate 320
precursors. The volatile thiols 3MH and 4MMP are influential odorants for a wide range of 321
wines (35). Since we have demonstrated that Str3p displays a broad specificity towards 322
cysteine-S-conjugates, we asked whether Str3p would also release the aromatic thiols 3MH 323
and 4MMP from their respective cysteinylated precursors, Cys-3MH and Cys-4MMP. S. 324
cerevisiae Str3p was able to release 12.3 µM of 4MMP, and 2.1 µM of 3MH, from 2 mM of 325
their respective precursors, when reactions were conducted with 31 µg/ml purified enzyme 326
(Fig. 3). This side activity of Str3p against Cys-4MMP and Cys-3MH corresponds to 1.3% 327
and 0.2% of the E. coli tryptophanase activity, respectively, and approximately 0.6-0.1% of 328
the specific activity towards its physiological substrate, L-cystathionine. In addition, a 329
reaction at pH 7.0 released 47% of the 4MMP released by Str3p at pH 7.5 (Fig. 3B). This 330
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reduced activity at pH 7.0 is consistent with the pH-rate profile of Str3p with L-cystathionine 331
as substrate (Fig. 2). The amount of 3MH and 4MMP formed was dependent on the 332
concentration of precursor for both enzymes. Cys-3MH was, however, the most effective 333
substrate at a concentration of 0.25 mM (0.54 vs 0.30 µM of free 3MH and 4MMP, 334
respectively). This led us to investigate the influence of Str3p in 3MH release under 335
fermentation conditions. 336
Volatile thiol release during fermentation of a S. Blanc grape must. Since Str3p 337
displayed a modest side activity towards the cysteinylated aroma precursors, we tested 338
whether overexpression of the STR3 gene in the wine yeast VIN13 could increase the release 339
of the aromatic thiol 3MH under oenological conditions. The modified strain, VIN13 (STR3), 340
was used to ferment a S. Blanc grape juice, alongside the VIN13 (CSL1) strain, previously 341
engineered to express the E. coli tryptophanase gene tnaA (28). The level of the STR3 mRNA 342
transcript was monitored during fermentation of a S. Blanc grape must by qRT-PCR at day 5 343
(when 60% of the sugar was consumed), and at day 15 (at the end of fermentation). In the 344
VIN13 control strain, the level of STR3 transcript under the control of its native promoter 345
increased 12.3-fold between day 5 and 15 of fermentation (Fig. 4). When compared with the 346
control strain, STR3 transcript levels in the modified strain, VIN13 (STR3), were 18-fold 347
higher at day 5, but only 50% higher by end of fermentation. The STR3 overexpression strain 348
displayed a sustained induction during the course of fermentation, whereas STR3 under the 349
control of the native promoter was highly expressed towards the end of fermentation. 350
All ferments proceeded at a similar rate, and were dry (less than 2 g/l sugar) after 15 351
days. Other chemical parameters (pH, glycerol, ethanol, and organic acid production) were 352
analyzed after fermentation with the different strains and found to be almost identical (Table 353
2), including concentrations of other key low molecular weight volatile sulfur compounds 354
known to adversely affect wine flavor. 355
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The VIN13 (CSL1) strain released substantial amounts of 3MH, 9.5 times more than 356
the control VIN13 strain, consistent with that described previously in a synthetic media 357
spiked with the Cys-3MH precursor (28). The 3MH concentration after fermentation with the 358
VIN13 (STR3) strain was 27% (278 ng/l) higher than in ferments with the VIN13 control 359
strain (p = 0.014) (Table 3). 360
DISCUSSION 361
The biochemical properties of the S. cerevisiae STR3 gene product have not 362
previously been characterized. Its function has been inferred by gene disruption, yielding a 363
yeast strain that could not grow on cystathionine as the sole sulfur source (3, 12). In this 364
study, we purified Str3p in order to determine some of its biochemical properties. We 365
confirmed that Str3p is a CBL, with the highest catalytic efficiency for L-cystathionine. In 366
accordance with previously characterized CBL enzymes, Str3p forms a stable tetrameric 367
enzyme consisting of four PLP-bound subunits. 368
As observed for enzymes of this class from A. thaliana, S. oleracea, and E. coli, Str3p 369
also displayed broad substrate specificity, including some activity towards the cysteine-S-370
conjugate substrates S-ethyl-L-cysteine, and S-methyl-L-cysteine. The latter substrate, 371
together with S-methyl-L-cysteine sulfoxide, occurs in high concentrations in Brassica and 372
Allium vegetables. Characteristic flavors of these vegetables are partly derived through 373
enzymatic degradation of these amino acids by CS lyases when their tissue is disrupted (11). 374
The in-vitro incubation of purified Str3p with cysteine-S-conjugates of 3MH and 375
4MMP confirmed our hypothesis that the enzyme has a residual cysteine-S-conjugate β-lyase 376
activity, and was able to cleave these substrates to release the corresponding aromatic thiols. 377
The reactions occurred in a concentration dependent manner, and Str3p displayed a 378
preference for Cys-3MH at low substrate concentrations, and for Cys-4MMP at high 379
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concentrations. To our knowledge, this is the first direct evidence of a purified yeast enzyme 380
displaying a CS β-lyase activity necessary to cleave the cysteine-S-conjugates of 4MMP and 381
3MH. 382
Previous studies based on gene disruption have identified several yeast genes (IRC7, 383
CYS3 and BNA3) that may contribute to the release of 3MH and/or 4MMP (13, 15, 30). The 384
contribution of each was, however unclear, since the effect of the deletions on thiol release 385
was strongly dependent on the experimental conditions used, and some results were 386
contradictory. Interestingly, IRC7, which was suggested encode the main enzymatic activity 387
involved in the release of 4MMP during fermentation (30), is annotated as a putative CBL 388
based on sequence similarity. This activity has never been demonstrated experimentally; 389
nonetheless, yeast CBLs apart from their physiological function of cleaving cystathionine to 390
yield homocysteine for methionine biosynthesis, could be harnessed to drive the formation of 391
aromatic thiols during beverage fermentation. We, therefore, integrated an additional copy of 392
the gene under the control of the constitutive promoter, PGK1P, in the commercial yeast 393
strain VIN13. Enhanced 3MH released by the VIN13 (STR3) strain represents the first proof-394
of-concept that a yeast-derived gene can be used in place of the CSL1 construct (28) to 395
harness latent flavor potential during wine fermentation. STR3 may not encode the optimal 396
enzyme for this purpose, since in addition to its side-activity against aromatic thiol 397
precursors, the STR3 gene is amongst a group of genes transcriptionally up-regulated during 398
fermentation (19). Nonetheless, since the VIN13 (STR3) strain was able release 278 ng/l 399
more 3MH then a control strain, and 3MH has a sensory detection threshold of 60 ng/l (32), 400
this increase illustrates the potential for CBLs to modulate wine aroma. Although the results 401
of the in-vitro experiment indicate that enzymatic activity of Str3p is directly responsible for 402
the increase in 3MH during wine fermentation, we can’t rule out that Str3p overexpression 403
could affect expression of other genes in sulfur amino acid metabolism. However, 404
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overexpression of Str3p didn’t affect the concentrations of other low molecular weight 405
volatile sulfur compounds known to adversely affect wine flavour. 406
In conclusion, we have demonstrated that a yeast enzyme, Str3p, is a CBL with side-407
activity towards cysteine-S-conjugated thiols, and that expression of STR3 can be 408
manipulated in wine yeast to effectively alter the composition of volatile thiols in a wine 409
fermentation. In-vitro characterization of other yeast enzymes with putative cysteine-S-410
conjugate β-lyase activity, in conjunction with structural bioinformatics, represents a path 411
forward to improve our understanding of volatile thiol release during fermentation, and 412
develop optimal self-cloned yeast strains for enhanced wine flavor. 413
414
415
ACKNOWLEDGEMENTS 416
The authors would like to thank the Department of Wine Innovation at Chr-Hansen 417
A/S, Denmark, and Dr. Hentie Swiegers for financial support. We would also like to 418
acknowledge Drew Sutton at Flinders University for assistance with size exclusion 419
chromatography and Dr. Paul Chambers for critical reading of the manuscript. Research at 420
the Australian Wine Research Institute is supported by Australia’s grapegrowers and 421
winemakers through their investment body, The Grape and Wine Research Development 422
Corporation, with matching funding from the Australian Government. 423
424
REFERENCES 425
1. Ausubel F. M., R. Brent, and R.E. Kingston (eds). 1994. Current Protocols in 426
Molecular Biology. Wiley, New York, NY. 427
on January 14, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
19
2. Bailly, S., V. Jerkovic, J. Marchand-Brynaert, and S. Collin. 2006. Aroma extraction 428
dilution analysis of Sauternes wines. Key role of polyfunctional thiols. J. Agric. Food 429
Chem. 54:7227-7234. 430
3. Barreto, L., A. Garcerá, K. Jansson, P. Sunnerhagen, and E. Herrero. 2006. A 431
peroxisomal glutathione transferase of Saccharomyces cerevisiae is functionally related 432
to sulfur amino acid metabolism. Eukaryotic Cell 5:1748-1759. 433
4. Novo, M., F. Bigey, E. Beyne, Virginie Galeote, F. Gavory, S. Mallet, B. Cambon, J. 434
Legras, P. Wincker, S. Casaregola, and S. Dequin. 2009. Eukaryote-to-eukaryote gene 435
transfer events revealed by the genome sequence of the wine yeast Saccharomyces 436
cerevisiae EC1118. Proc. Natl. Acad. Sci. 106:16333-16338 437
5. Bouchilloux, P., P. Darriet, R. Henry, V. Lavigne-Cruège, and D. Dubourdieu. 1998. 438
Identification of volatile and powerful odorous thiols in Bordeaux red wine varieties. J. 439
Agric. Food Chem. 46:3095-3099. 440
6. Breitinger, U., T. Clausen, S. Ehlert, R. Huber, B. Laber, F. Schmidt, E. Pohl, and 441
A. Messerschmidt. 2001. The three-dimensional structure of cystathionine β-lyase from 442
Arabidopsis and its substrate specificity. Plant Physiol. 126:631-642. 443
7. Clausen, T., R. Huber, B. Laber, H. D. Pohlenz, and A. Messerschmidt. 1996. Crystal 444
structure of the pyridoxal-5'-phosphate dependent cystathionine β-lyase from Escherichia 445
coli at 1.83 Å. J. Mol. Biol. 262:202-224. 446
8. Ejim, L. J., J. E. Blanchard, K. P. Koteva, R. Sumerfield, N. H. Elowe, J. D. 447
Chechetto, E. D. Brown, M. S. Junop, and G. D. Wright. 2007. Inhibitors of bacterial 448
cystathionine β-lyase: leads for new antimicrobial agents and probes of enzyme structure 449
and function. J. Med. Chem. 50:755-764. 450
on January 14, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
20
9. Ejim, L. J., V. M. D’Costa, N. H. Elowe, J. C. Loredo-Osti, D. Malo, and G. D. 451
Wright. 2004. Cystathionine β-lyase is important for virulence of Salmonella enterica 452
Serovar Typhimurium. Infect. Immun. 72:3310-3314. 453
10. Gentry-Weeks, C. R., J. M. Keith, and J. Thompson. 1993. Toxicity of Bordetella 454
avium β-cystathionase toward MC3T3-E1 osteogenic cells. J. Biol. Chem. 268:7298-455
7314. 456
11. Hamamoto, A., and M. Mazelis. 1986. The C-S Lyases of Higher Plants: Isolation and 457
properties of homogeneous cystine lyase from Broccoli (Brassica oleracea var botrytis) 458
buds. Plant Physiol. 80:702-706. 459
12. Hansen, J., and P. F. Johannesen. 2000. Cysteine is essential for transcriptional 460
regulation of the sulfur assimilation genes in Saccharomyces cerevisiae. Mol. Gen. Genet. 461
263:535-542. 462
13. Harsch, M. 2009. Ph.D. thesis. University of Auckland, New Zealand. Identification of 463
yeast genes involved in Sauvignon Blanc aroma development. 464
14. Howell, K. S., J. H. Swiegers, G. M. Elsey, T. E. Siebert, E. J. Bartowsky, G. H. 465
Fleet, I. S. Pretorius, and M. A. de Barros Lopes. 2004. Variation in 4-mercapto-4-466
methyl-pentan-2-one release by Saccharomyces cerevisiae commercial wine strains. 467
FEMS Microbiol. Lett. 240:125-129. 468
15. Howell, K. S., M. Klein, J. H. Swiegers, Y. Hayasaka, G. M. Elsey, G. H. Fleet, P. B. 469
Høj, I. S. Pretorius, and M. A de Barros Lopes. 2005. Genetic determinants of volatile-470
thiol release by Saccharomyces cerevisiae during wine fermentation. Appl. Environ. 471
Microbiol. 71:5420-5426. 472
16. Irmler, S., S. Raboud, B. Beisert, D. Rauhut, and H. Berthoud. 2008. Cloning and 473
characterization of two Lactobacillus casei genes encoding a cystathionine lyase. Appl. 474
Environ. Microbiol. 74:99-106. 475
on January 14, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
21
17. King, E.S., P. Osidacz, C. Curtin, S. Bastian, and I. L. Francis. Austr. J. Grape Wine 476
Res., in press. Assessing desirable levels of sensory properties in Sauvignon Blanc wines 477
– consumer preferences and contribution of key aroma compounds. 478
18. Kotseridis, Y., J. L. Ray, C. Augier, and R. Baumes. 2000. Quantitative determination 479
of sulfur containing wine odorants at sub-ppb levels. 1. Synthesis of the deuterated 480
analogues. J. Agric. Food Chem. 48:5819-5823. 481
19. Marks, V. D., S. J. Ho Sui, D. Erasmus, G. K. van der Merwe, J. Brumm, W. W. 482
Wasserman, J. Bryan, and H. J. J. van Vuuren. 2008. Dynamics of the yeast 483
transcriptome during wine fermentation reveals a novel fermentation stress response. 484
FEMS Yeast Res. 8:35-52. 485
20. Martínez-Cuesta, M. C., C. Peláez, J. Eagles, M. J. Gasson, T. Requena, and S. B. 486
Hanniffy. 2006. YtjE from Lactococcus lactis IL1403 is a C-S lyase with α, γ-elimination 487
activity toward methionine. Appl. Environ. Microbiol. 72:4878-4884. 488
21. Pardon, K. H., S. D. Graney, D. L. Capone, J. H. Swiegers, M. A. Sefton, and G. M. 489
Elsey. 2008. Synthesis of the individual diastereomers of the cysteine conjugate of 3-490
mercaptohexanol (3-MH). J. Agric. Food Chem. 56:3758-3763. 491
22. Ravanel, S., D. Job, and R. Douce. 1996. Purification and properties of cystathionine β-492
lyase from Arabidopsis thaliana overexpressed in Escherichia coli. Biochem. J. 320:383-493
392. 494
23. Ringquist, S., S. Shinedling, D. Barrick, L. Green, J. Binkley, G.D. Stormo, and L. 495
Gold. 1992. Translation initiation in Escherichia coli: sequences within the ribosome-496
binding site. Mol. Microbiol. 6:1219-1229. 497
24. Schneider, G., H. Käck, and Y. Lindqvist. 2000. The manifold of vitamin B6 498
dependent enzymes. Structure 8:R1-R6. 499
on January 14, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
22
25. Shinkaruk, S., Thibon, C., Schmitter, J., Babin, P., Tominaga, T., Degueil, M., 500
Desbat, B., Jussier, C., Bennetau, B., Dubourdieu, D., Bennetau-Pelissero, C. 2008. 501
Surprising Structral Lability of a Cystein-S-Conjugate Precursor of 4-Methyl-4-502
sulfanylpentan-2-one, a Varietal Aroma in Wine of Vitis vinifera L. Cv. Sauvignon Blanc. 503
Chem. Biodiv. 5: 793-810 504
26. Siebert, E. T., Solomon M. R., Pollnitz, A. P. and Jeffery, D. W. 2010. Selective 505
determination of volatile sulfur compounds in wine by gas chromatography with sulphur 506
chemiluminescence detection. J. Agric. Food Chem. 58: 9454-9462 507
27. Staton, A. L., and M. Mazelis. 1991. The C-S lyases of higher plants: homogeneous 508
beta-cystathionase of spinach leaves. Arch. Biochem. Biophys. 290:46-50. 509
28. Swiegers, J. H., D. L. Capone, K. H. Pardon, G. M. Elsey, M. A. Sefton, I. L. 510
Francis, and I. S. Pretorius. 2007. Engineering volatile thiol release in Saccharomyces 511
cerevisiae for improved wine aroma. Yeast 24:561-574. 512
29. Swiegers, J. H., R. L. Kievit, T. Siebert, K. A. Lattey, B. R. Bramley, I. L. Francis, E. 513
S. King, and I. S. Pretorius. 2009. The influence of yeast on the aroma of Sauvignon 514
Blanc wine. Food Microbiol. 26:204-211. 515
30. Thibon, C., P. Marullo, O. Claisse, C. Cullin, D. Dubourdieu, and T. Tominaga. 516
2008. Nitrogen catabolic repression controls the release of volatile thiols by 517
Saccharomyces cerevisiae during wine fermentation. FEMS Yeast Res. 8:1076-1086. 518
31. Thomas, D., and Y. Surdin-Kerjan. 1997. Metabolism of sulfur amino acids in 519
Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 61:503-532. 520
32. Tominaga, T., A. Furrer, R. Henry, and D. Dubourdieu. 1998. Identification of new 521
volatile thiols in the aroma of Vitis vinifera L. var. Sauvignon blanc wines. Flavour Fragr. 522
J. 13:159-162. 523
on January 14, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
23
33. Tominaga, T., C. Peyrot des Gachons, and D. Dubourdieu. 1998. A new type of flavor 524
precursors in Vitis vinifera L. cv. Sauvignon blanc: S-Cysteine Conjugates. J. Agric. Food 525
Chem. 46:5215-5219. 526
34. Tominaga, T., G. Guimbertau, and D. Dubourdieu. 2003. Role of certain volatile 527
thiols in the bouquet of aged champagne wines. J. Agric. Food Chem. 51:1016-1020. 528
35. Tominaga, T., I. Masneuf, and D. Dubourdieu. 2004. Powerful aromatic volatile thiols 529
in wines made from several Vitis vinifera grape varieties and their releasing mechanism. 530
ACS Symp. Ser. 871:314-337. 531
36. Troccaz, M., F. Benattia, G. Borchard, and A. J. Clark. 2008. Properties of 532
recombinant Staphylococcus haemolyticus cystathionine β-lyase (metC) and its potential 533
role in the generation of volatile thiols in axillary malodor. Chem. Biodivers. 5:2372-534
2385. 535
37. Vermeulen, C., I. Lejeune, T. T. H. Tran, and S. Collin. 2006. Occurrence of 536
polyfunctional thiols in fresh lager beers. J. Agric. Food Chem. 54:5061-5068. 537
38. Vermeulen, C., L. Gijs, and S. Collin. 2005. Sensorial contribution and formation 538
pathways of thiols in foods: a review. Food Rev. Int. 21:69-145. 539
39. Wakabayashi, H., M. Wakabayashi, W. Eisenreich, and K. Engel. 2004. 540
Stereochemical course of the generation of 3-mercaptohexanal and 3-mercaptohexanol by 541
β-lyase-catalyzed cleavage of cysteine conjugates. J. Agric. Food Chem. 52:110-116. 542
40. Wu, W., and D. R. Morris. 1973. Biosynthetic arginine decarboxylase from Escherichia 543
coli. Subunit interactions and the role of magnesium ion. J. Biol. Chem. 248:1696-1699 544
41. Yoshida, Y., M. Negishi, A. Amano, T. Oho, and Y. Nakano. 2003. Differences in the 545
βC-S lyase activities of viridans group streptococci. Biochem. Biophys. Res. Commun. 546
300:55-60. 547
548
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TABLES 549
550
TABLE 1. Substrate specificity of purified Str3p
Substrate Relative activity (%)a
L-cystathionineb 100 ± 10.9
L-djenkolate 154 ± 11.0
L-cystine 22.0 ± 2.1
S-ethyl-L-cysteine 9.0 ± 0.7
S-methyl-L-cysteine 7.4 ± 0.3
L-cysteine 1.0 ± 0.3
L-methionine 0.0
a The formation of pyruvate was detected by HPLC and
expressed relative to the activity with L-cystathionine ± SD.
Data are from triplicate experiments.
b Specific activity towards L-cystathionine was 1258 ± 138
µmol/min/mg protein.
551
552
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553
554
555
556
557
558
TABLE 2. Basic composition for the Sauvignon Blanc wines made using
the modified VIN13 (STR3), VIN13 (CSL1), and the control VIN13 strains
Units VIN13 VIN13
(STR3)
VIN13
(CSL1)
Alcohol % (v/v)
a
11.5 ± 0.3 11.6 ± 0.2 11.4 ± 0.3
Residual sugar g/l a
0.1 ± 0.0 0.4 ± 0.2 0.0 ± 0.0
Acetic acid g/l a
0.05 ± 0.01 0.06 ± 0.01 0.03 ± 0.005
Glycerol g/l a
4.6 ± 0.13 4.4 ± 0.03 4.5 ± 0.31
Malic acid g/l a
2.6 ± 0.02 2.5 ± 0.01 2.5 ± 0.05
Tartaric acid g/l a
1.8 ± 0.01 1.8 ± 0.01 1.8 ± 0.12
Succinic acid g/l a
2.0 ± 0.07 2.0 ± 0.04 2.0 ± 0.14
Hydrogen sulfide µg/l b 1.2 ± 0.2 1.2 ± 0.3 1.6 ± 1.1
Methanethiol µg/l b 4.1 ± 1.8 5.0 ± 1.1 4.6 ± 2.4
Ethanethiol µg/l b nd
c nd nd
Dimethyl sulfide µg/l b 11.4 ± 6.0 10.7 ± 3.2 10.6 ± 5.7
Carbon disulfide µg/l b 0.3 ± 0.1 0.4 ± 0.1 0.5 ± 0.2
Diethyl sulfide µg/l b 0.3 ± 0.0 0.3 ± 0.0 0.2 ± 0.0
Methyl thioacetate µg/l b 3.2 ± 0.8 4.6 ± 2.6 2.9 ± 1.0
Dimethyl disulfide µg/l b nd nd nd
Ethyl thioacetate µg/l b 0.3 ± 0.4 0.5 ± 0.6 0.5 ± 0.5
Diethyl disulfide µg/l b nd nd nd
Data are from triplicate fermentations ± SD by a HPLC and
b GC/SCD.
c Not detected
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559
TABLE 3. Production of 3MH in a Sauvignon
Blanc grape must using the modified VIN13
(STR3), VIN13 (CSL1), and the control VIN13
strains.
3MH (ng/l) a t-testb
VIN13 1084 ± 66
VIN13 (STR3) 1362 ± 84 0.014
VIN13 (CSL1) 10268 ± 548 0.001
a Data shown are the mean of triplicate
fermentations ± SD quantified by headspace
GC/MS.
b One-tailed student t-test.
560
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561
FIGURES AND FIGURE LEGENDS 562
563
564
FIG. 1. SDS-polyacrylamide gel of
nickel affinity purified Str3p. Lane 1:
cleared lysate; lane 2: flow through;
lane 3: 10 mM imidazole wash; lane 4:
100 mM imidazole wash; lane 5: elution
with 500 mM imidazole.
565
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566
567
568
569
570
571
572
573
FIG. 2. The activity of Str3p towards L-cystathionine was measured by detection 574
of pyruvate by HPLC. The activity was normalised against a blank, and expressed as 575
percentage compared to the maximal activity at pH 8.75. Data shown are the mean of three 576
experiments. The SD did not exceed 10% in any of the values. 577
578
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579
580
581
582
583
584
FIG. 3. GC/MS quantification of enzymatic reactions with purified Str3p. The release of 3MH and
4MMP was quantified with headspace GC/MS in reactions incubated with 0.25 mM or 2 mM cysteine-
S-conjugated precursor. Experiments were carried out with 31 µg/ml purified Str3p at 28°C and at pH
pH 7.5 to minimize hydrolysis of the 4MMP precursor (25). Data shown are the mean of triplicate
experiments ± SD of Str3p reactions and empty-vector controls (pET), which were significantly
different (p < 0.01) for both substrates. An additional negative control, using heat-inactivated Str3p,
was indistinguishable from the empty-vector control (p = 0.225 for 3MH, and p = 0.442 for 4MMP).
We observed a strong correlation (R2 = 0.95) between thiol and pyruvate formation for both Cys-3MH
and Cys-4MMP (data not shown). Hatched bars show experiment carried out at pH 7.0
585
b) a)
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586
587
588
589
FIG. 4. Quantitative RT-PCR of the STR3 mRNA level at day 5 (white) and day 15 (light 590
grey) during fermentation with the commercial wine yeast, VIN13, and a strain modified to 591
overexpress STR3, VIN13(STR3). Data shown are the mean of 4 data points from duplicate 592
fermentations ± SEM. 593
594
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